The invention relates to a method and a device for matrix-assisted laser desorption ionization.
Conventional methods for the ionization of substances for analysis by mass spectrometry, where a solid substance is heated, for example, transferred to the gaseous phase and ionized there by electron collision, cannot be applied to the large organic and biological molecules. Electron collision with energetic electrons (typically 70 eV) leads to a substantial fragmentation of this species, whereby only small fragments can be observed. On the other hand, even if energy is only supplied at a slow rate, as is always the case when heating a solid sample, large organic molecules decompose before they can vaporize. Only if energy supply takes place at an extremely fast rate, as is the case with a laser beam, for example, the usually slower decomposition process of the molecules does not occur at all.
Laser desorption ionization was already used in the last decade to transfer large organic molecules to the gaseous phase and to ionize them. A special type of laser desorption ionization (LDI) is matrix-assisted laser desorption ionization (MALDI). The detailed review article by F. Hillenkamp, M. Karas, R. Beavis, and B. Chait in xe2x80x9cAnalytical Chemistryxe2x80x9d, Volume 63, year 1991, on pages 1193A-1203A, reports about this technology. In MALDI the analyte molecules are mixed with a so-called matrix. The analyte/matrix molar ratio is 1:102 to 1:104. The laser energy is absorbed by matrix molecules and passed on to analyte molecules. The latter thus receive the necessary energy to enter the gaseous phase and are thereby partially ionized. The ionization usually takes place by a protonation. The substances which are mostly used as a matrix are proton donors. In special cases, alkali-metal salts or silver salts are also added to achieve alkali-metal or silver attachment. With some samples both protonated analyte molecules and small quantities of sodium adducts are also observed. The latter often form due to the presence of sodium chloride residues in biological samples.
Experience shows that the ions formed by the MALDI process have kinetic energy which is not negligible and which can be up to or over 10 eV. Since in the classical time-of-flight mass spectrometry the MALDI-generated ions are normally extracted and accelerated at voltages of between 15 and 30 kV, an energy spread of about 10 eV is relatively unimportant here. However, in ion trap mass spectrometers like the Fourier transform ion cyclotron resonance mass spectrometers (FTICRs) the ions produced in an external ion source must be transferred to the trap and captured there. Therefore, the extraction of the MALDI-generated ions no longer takes place here at a potential difference of several kilovolts. However, in the range of low ion extraction potential, which does not exceed 10-20 V, a fluctuation of excess energy in the region of 10 eV is too high and therefore causes enormous difficulties. It leads to a considerable intensity variation of the obtained mass signals and therefore to irreproducible analytical results.
In Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) one attempts by various methods to capture the ions in the trap with as few losses as possible and first of all effectively reduce their energy for the ICR measurements. This happens, for instance, by dynamic trapping, where an inert gas is also pulsed into the ion cyclotron resonance trap (ICR trap) in order to absorb the kinetic energy of the ions by collisions with gas molecules or atoms.
One can also attempt to cool the ions in the ion source. This method uses an increased static pressure in the source so that the ions generated by MALDI lose their energy immediately by collisions. According to a different method the MALDI process can even be performed at atmospheric pressure. The U.S. Pat. No. 5,663,561 describes such a device for atmospheric pressure laser desorption ionization. In this case matrix substances are used which undergo photolytic of thermolytic decomposition. However, as opposed to MALDI, the gases formed during this atmospheric pressure desorption are not intended to ionize the large analyte molecules. The selection of matrix molecules therefore only depends on their capability to liberate the large molecules by desorption. Analyte molecules catapulted into the gaseous phase are then ionized for example by a corona discharge. The corona discharge primarily forms nitrogen ions which, in turn, ionize water molecules in moist air, which then perform the ionization of the analyte molecules.
In FTICR mass spectrometry with an external MALDI ion source the dynamic trapping of the ions that are formed in a low voltage MALDI source requires an electrical xe2x80x9copening and closingxe2x80x9d of the ICR trapping plate facing the ion source. This is usually combined with an increase of the trapping potential of the rear trapping plate. Capturing higher-energy ions in an ion trap is always problematic. A loss-free capture is especially difficult if a swarm of ions with a broad energy spread arrives from such a MALDI source. In FTICR mass spectrometry one also uses frequently a pulsed inert gas in the ICR trap. Collisions with these gas molecules remove the excess energy of the MALDI-generated ions. Thus, reduced-energy ions are obtained which can be resonantly excited and detected in the ICR trap. However, FTICR mass spectrometry requires a very good vacuum of around xe2x89xa610xe2x88x929 mbar in the analyzer range, particularly in order to achieve the high resolution. In FTICR one avoids operating at pressures above 10xe2x88x928 mbar since the broadening of the ion cyclotron resonance signals disturbs the measurements. If the capture of the ions is associated with a gas pulse, one has to wait for a time period after each gas pulse-assisted ion trapping until the pulsed gas is pumped out. This time period can be 5-10 seconds or even longer, thus, a much longer time is required to add up a multitude of spectra.
These problems appear if the ions in MALDI source are transferred to the ICR trap by a low voltage extraction and acceleration. Therefore, it seems to be simpler to absorb the excess kinetic energy of the MALDI-generated ions already in the source by collisions with gas molecules.
The alternative to increasing the pressure in the ICR trap is to statically increase the pressure in the ion source of an FTICR mass spectrometer. As described above, the collisions with gas molecules would already remove the excess energy from MALDI-generated ions at the location of their formation. A MALDI ion source with statically increased pressure, in connection with time-of-flight mass spectrometry though, was described in the publication by A. N. Krutchinsky, A. V. Loboda, V. L. Spicer, R. Dworschak, W. Ens, and K. G. Standing in xe2x80x9cRapid Communications in Mass Spectrometryxe2x80x9d, Volume 12, year 1998, on pages 508 to 518. However, when a statically increased source pressure is applied to FTICR mass spectrometry in order to achieve a higher analyte yield for MALDI, it leads also to a higher static pressure in the ICR trap. A certain increase in trap pressure occurs despite a differentially pumped system if the source pressure rises to levels such as 0.01 or 0.1 mbar, which in turn can considerably affect the performance of the FTICR system (broader peak, reduced resolution).
Ultimately, the methods proposed so far using a high static pressure in the laser desorption ion source, e.g. atmospheric pressure (4 or 5 orders of magnitude higher than a source pressure increased statically to 0.1 or 0.01 mbar) are not intended for classic MALDI processes. They are new techniques associated with atmospheric pressure ionization with the aid of an additional reactant gas, whereby the classic matrix substances are not normally used.
It is the objective of the invention to find a device and a method for absorbing the excess kinetic energy of the ions formed in a MALDI ion source, immediately after their formation and locally in the ion source. During the cooling process of ions, the vacuum in the rest of the mass spectrometer should preferably not be affected.
The invention consists of absorbing the excess kinetic energy of the MALDI-generated ions in the ion source by collisions with the introduced gas molecules. The invention introduces a device, in which the collision gases are pulsed directly onto the MALDI sample through a thin tube or a capillary by using a pulse valve. A gas pulse which is synchronized with the laser pulse (a certain period of time before or after the laser pulse, or during the laser pulse) generates a short-time increase of the local pressure directly on the surface of the applied substance. The analyte ions generated with excess energy collide with the pulsed gas molecules and lose some of their energy before they leave the ion source. These ions can now be extracted from the ion source zone at a low voltage (e.g. 10-20 volts) and passed on into the mass spectrometry analyzer. If the excess energy is absorbed effectively, other problems associated with a spread in the ion energy are also eliminated. The local pressure increase on the surface of the sample can be achieved by using much less collision gas than a static pressure increase of the source would require. The effect is the same but a much smaller quantity of collision gas is required and therefore does not cause an unnecessary pressure increase in the rest of the vacuum system of the mass spectrometer, which could lead to a performance reduction.
A different perspective of the invention is that reaction gases can be introduced through other gas tubes to the direct vicinity of the substance to be desorbed. In this way primary ions which form by matrix assisted laser desorption can react with reactive neutral molecules. Product ions from these reactions are transferred into the mass spectrometric analyzer just like the desorbed primary ions and detected. Ion molecule reactions in a MALDI ion source are interesting not only with regard to the ion chemistry. They can also be useful for analytical purposes. If a suitable reaction gas is selected, such reactions of the desorbed ions can lead to a kind of xe2x80x9cderivatization effectxe2x80x9d and therefore provide a further dimension of information for the substance to be analyzed.
Observations indicate that by a MALDI process at increased pressure (due to inert gas) more analyte ions can be detected near the point of desorption. It may be assumed that the collisionally induced cooling of the desorbed ions, which otherwise have excess energy, prevents losses during collection and transmission of the ions. The slower expansion of the desorbed swarm of matrix and analyte ions, as well as matrix and analyte molecules, may also permit greater production of analyte ions in the source, because there is more time available for post desorptive ion-molecule interactions (e.g. proton transfer).
An RF multipole ion guide which is placed directly in front of the laser desorption point can additionally protect a swarm of ions against fast expansion. When the MALDI process is assisted by an inert gas pulse and the ions are also simultaneously collected in a multipole ion guide direct, the above-mentioned effects are intensified. Here the multipole ion guide can, for example, be a quadrupole, a hexapole or an octopole.